A. Field of the Invention
This invention relates generally to an air matrix material for use in chemical reactions to compartmentalize reagents and/or reaction products of such chemical reactions which are amenable to subsequent process or reaction steps or transfer steps. Specifically, this invention relates to the method of synthesizing multiple copies of a target nucleic acid sequence within an air matrix material in order to reduce the loss of reagents or reaction solutions associated with these amplification reactions and more particularly to reduce from the test container the aerosolation of amplification products produced from these reactions.
B. Technical Disclosure
When an abundant supply of starting materials is available, nucleic acid target sequences are readily detectable by common hybridization techniques following extraction. Several sensitive amplification techniques have been developed to allow detection of target sequences from very small amounts of starting material. The most notable of these methods include Polymerase Chain Reaction (PCR), Transcription Mediated Amplification (TMA), and Nucleic Acid Sequence Based Amplification (NASBA). These techniques produce either a logarithmic or exponential increase in the number of target sequences or amplicons. Out of all of these methods, the most widely used method is PCR, which uses primer-dependent synthesis of complementary nucleic acids by thermally stable DNA polymerase to produce copies of the target sequence. Repeated cycling between the optimal temperatures for annealing of the primers, extension by the polymerase and denaturation of the duplex nucleic acids produces an exponential accumulation of copies of the original target sequence. Usually this repeated cycling is carried out in stoppered plastic tubes, such as Eppendorf.RTM. tubes or other appropriate containers. Once the desired number of amplicons have been produced during cycling, these stoppered tubes are reopened for fluid transfer. It is during this act of unstoppering the reaction tubes and transferring of reaction fluids or products that aerosols may be created and released. These aerosols which are then dispersed into the environment can contain amplicons such as DNA which can easily contaminate other reactions.
Another problem presented in these and other reactions is retaining reagents, reaction solutions, and reaction products in a definable space for chemical reactions in general, particularly where there is some risk of contamination from dispersal of such products.
More recently, TMA and NASBA were developed that make possible the exponential amplification of single stranded nucleic acid targets at a single temperature. Both techniques involve the incorporation of an RNA polymerase promoter sequence into the target nucleic acid sequence. This promoter subsequently directs transcription by an RNA polymerase to make multiple RNA copies of the original target sequence. Using reverse transcriptase and RNase H activities, each amplicon can serve as a target molecule for subsequent amplification, allowing the reaction to cycle at a constant temperature.
In theory, the use of amplification techniques allows for the detection of a single target molecule, although more practical estimates suggest that ten to one or several hundred molecules are routinely necessary in a test sample. While it is the intent of each of the above techniques to increase the amount of the target sequence or amplicon by many orders of magnitude, the abundance of such reaction products can be problematic if they are not retained in definable space.
Equally important in performing these types of reactions is maintaining the proper activity and volume requirements for subsequent identification and detection procedures; since amplicons, reagents, and reaction solutions can be easily lost or aerosolized, by transfer steps. The loss of aerosols from the reaction solution can easily contain up to one million copies of molecules, each of which can act as a target molecule for an unintentional amplification and the loss of volume of reagents and reaction solutions may negatively affect the test process.
To deal with this contamination problem, laboratories have tried a variety of measures. These measures include stringent cleaning with caustic reagents such as sodium hypochlorite in between each run of an amplification reaction or of maintaining separate work sites for setting up and for running the reactions.
The present invention addresses the problem(s) by demonstrating that nucleic acid amplification can occur within the matrix of a porous, absorbent material, such as foam rubber or a sponge, which can reduce the spread of amplification products and the loss of reagents and reaction solutions outside the test vessel. This invention advantageously controls the unwanted loss of reagents and reaction solutions, as well as, the distribution of contaminating molecules. This invention also demonstrates that the use of an air matrix is compatible with amplification reactions.